Analysis of leukocyte membrane protein interactions using protein microarrays
© Letarte et al; licensee BioMed Central Ltd. 2005
Received: 08 November 2004
Accepted: 01 March 2005
Published: 01 March 2005
Protein microarrays represent an emerging class of proteomic tools to investigate multiple protein-protein interactions in parallel. A sufficient proportion of immobilized proteins must maintain an active conformation and an orientation that allows for the sensitive and specific detection of antibody and ligand binding. In order to establish protein array technology for the characterization of the weak interactions between leukocyte membrane proteins, we selected the human leukocyte membrane protein CD200 (OX2) and its cell surface receptor (hCD200R) as a model system. As antibody-antigen reactions are generally of higher affinity than receptor-ligand binding, we first analyzed the reactivity of monoclonal antibodies (mAb) to normal and mutant forms of immobilized CD200R.
Fluorescently labelled mAb DX147, DX136 and OX108 were specifically reactive with immobilized recombinant hCD200R extracellular region, over a range of 0.1–40 μg ml-1 corresponding to a limit of sensitivity of 0.01–0.05 femtomol per spot. Orientating hCD200R using capture antibodies, showed that DX147 reacts with an epitope spatially distinct from the more closely related DX136 and OX108 epitopes. A panel of soluble recombinant proteins with mutations in hCD200R domain 1 produced by transiently transfected cells, was arrayed directly without purification and screened for binding to the three mAb. Several showed decreased binding to the blocking mAb DX136 and OX108, suggesting close proximity of these epitopes to the CD200 binding site. Binding of hCD200 to directly immobilized rat, mouse, and hCD200R was achieved with multimeric ligands, in the form of biotinylated-hCD200 coupled to FITC-labelled avidin coated beads.
We have achieved sensitive, specific and reproducible detection of immobilized CD200R with different antibodies and mapped antigenic epitopes for two mAb in the vicinity of the ligand binding site using protein microarrays. We also detected CD200 binding to its receptor, a low affinity interaction, using beads presenting multivalent ligands. Our results demonstrate the quantitative aspects of protein arrays and their potential use in detecting simultaneously multiple protein-protein interactions and in particular the weak interactions found between leukocyte membrane proteins.
Protein-protein interactions are fundamental to biological processes and their analysis is essential for the understanding of cellular pathways. Given the complexity and the dynamic range of the proteome, estimated at 107 proteins, the elucidation of protein interactions requires the development of comprehensive, high-throughput proteomic methods that allow quantification of multiple proteins simultaneously [1, 2]. The development of protein microarrays represents an attractive new high-throughput technology platform. It involves the printing of ordered arrays of biomolecules onto a solid surface in miniaturized format that allows for the simultaneous determination of multiple interactions using small amounts of samples within a single experiment. The basic principles for highly sensitive "microspot" ligand-binding assays were described by Ekins [3, 4] who proposed the "ambient analyte theory" and showed that microspots containing small amounts of capture molecules were able to detect low analyte concentrations with very high accuracy and sensitivity. Since then, miniaturized protein arrays are emerging as one of the most powerful proteomics tools but their application is far more complex  than the DNA microarrays (reviewed in [6–8]) due to structural complexity and heterogeneity of proteins, including their post-translational modifications. Binding of the proteins onto the solid surface of an array must maintain tertiary structure sufficient for functions such as receptor-ligand binding or antibody reactivity. Chemically derivatized microarray surfaces [9, 10] or the use of mAb [11, 12] have been shown to maintain protein functionality, thus increasing the potential for successful application of microarray technology in proteomics.
The study of leukocyte membrane protein interactions provides a particular need because of the large number of interactions yet to be defined [13, 14] and a technical challenge as these interactions are often of very low affinity with KD in the range 1–200 μM [15, 16]. Although weak, these interactions are important in the context of leukocytes interacting with other cells as illustrated by all the functional data on the interaction of CD8 with MHC Class II (KD = 200 μM) . The proteins involved usually contain folded domains, the most common type belonging to the immunoglobulin superfamily (IgSF) . Such domains often interact through large faces of the proteins and require proper folding [18, 19]. When measuring low affinity interactions, misleading results can be obtained from unfolded or aggregated materials which are not really a problem when dealing with high affinity interactions such as with cytokines and their receptors, or between proteins and linear epitopes such as lectins and carbohydrates. In addition many leukocyte surface proteins are heavily glycosylated and the oligosaccharides, even if not directly involved in binding, may be important in maintaining biologically active proteins . Thus, in applying the protein microarray technology to the study of leukocyte surface protein interactions, it is imperative that the proteins are expressed in eukaryotic systems to ensure correct disulphide bond formation and post-translational modifications.
In this study we chose a well characterized interaction between CD200 (previously called OX2) and its receptor CD200R (reviewed in ) as a model system to devise a high throughput protein array method for characterization of the interactions between leukocyte surface proteins. CD200 is a widely distributed membrane protein with two extracellular IgSF domains and a short cytoplasmic region unlikely to signal. It interacts with a receptor (CD200R) expressed mostly on myeloid cells, which also has two extracellular IgSF domains but a longer cytoplasmic region with several tyrosine residues that can be phosphorylated . Functional analysis suggests that the leukocyte CD200 protein can mediate a down-regulatory signal to myeloid cells through the inhibitory CD200R. Thus the CD200 null mice have an increased susceptibility to autoimmune disease induction and myeloid cells expressing CD200R are more activated . CD200 and a viral homologue found in Kaposi sarcoma virus, when expressed at the cell surface, gave inhibition of production of inflammatory cytokines from activated macrophages ; and targeting the CD200-CD200R interaction with agonistic mAb or CD200-Fc fusion proteins in vivo ameliorates autoimmunity in disease models [25, 26].
Protein arrays can be divided into two major classes: 'forward phase' if the analytes are captured from solution; or 'reverse phase' if the analytes are bound directly to the solid phase . In forward phase protein microarrays, a bait molecule such as an antibody is immobilized onto a solid support to capture the analytes which can be proteins in purified form, or in complex solutions such as cell lysates  or tissue samples [27, 28]. The bound analytes are detected either by direct labelling or via a secondary antibody. In reverse phase arrays, the analytes (typically purified proteins or cell lysates) are directly immobilized on the solid phase and antibodies or interacting proteins are applied in solution phase. The analytes can be labelled directly or detected using tags and signal amplification.
We have used the forward phase approach in the mapping of antigenic epitopes of hCD200R where different antibodies were immobilized on epoxy coated glass slides, incubated with the hCD200R analyte and detected with fluorescently labelled anti-CD200R antibodies. We have applied reverse phase arrays to three different purposes: -to test the reactivity of the fluorescently labelled mAb with directly immobilized hCD200R protein, -to map epitopes located near the ligand binding site using arrayed mutant hCD200R recombinant proteins and detection with fluorescently labelled mAb that block ligand-receptor interactions and -to detect the low affinity binding of immobilized CD200R to the multivalent CD200 ligand presented on fluorescently labelled beads. Our study extends the use of protein microarrays to the detection of transient cell surface protein interactions, which are of lower affinity than the reported cytokine arrays [29, 30].
Results and discussion
Quantitative binding of DX147, DX136 and OX108 mAb to human CD200R
Orientation via antibody immobilization for epitope mapping on human CD200R
Mapping of CD200R antigenic epitopes using mutants
Both CD200R and its ligand, CD200 contain two extracellular IgSF domains. The ligand-receptor interaction is therefore likely to occur in an end-to-end topology, requiring opposing cell surfaces to come into close proximity . Previous studies have shown that the membrane distal N-terminal domain of CD200 is involved in binding its receptor . In a recently published study , site directed mutagenesis was employed to map the ligand-binding domain of human CD200R using the structure of a typical Ig V domain, that of the human junctional adhesion molecule 1, JAM1 to predict the positions of out-pointing residues . A panel of mutants was designed so as to target residues likely to be out-pointing from predictions of the beta strands of the N-terminal IgSF domain of hCD200R. The binding sites of CD200 and of the OX108 mAb known to block ligand interaction to the hCD200R mutants, were shown to be on the GFCC' face of the N-terminal IgSF domain .
hCD200R protein mutants tested. Mutant proteins were constructed as described in , expressed by transient transfection in serum-free medium, concentrated and immobilized on epoxy-derivatized slides. The predicted positions of the residues are located in the modelled V-like N-terminal IgSF domain unless noted otherwise (C domain).
D-E loop/ E strand
A strand C domain
A-B loop C domain
B-C loop C domain
Reactivity of human, rat and mouse CD200R with multimeric human CD200
In order to study interactions of leukocyte membrane proteins using high throughput microarray techniques, it was essential that the proteins be immobilized at low concentrations and in a biologically active form. It is critical that weak interactions between leukocyte membrane proteins be detected without interference by the anomalous binding due to denatured proteins, which is more of a contributing factor in the study of low affinity interactions. We first established that recombinant CD200R proteins could be immobilized directly in reverse phase arrays, in a conformation capable of reacting with three different mAb. We then demonstrated that recombinant mutant hCD200R proteins produced in transient expression systems were present in sufficient amounts to be immobilized directly and tested for reactivity with specific mAb, permitting mapping of epitopes. These data show that high throughput analysis of cell surface proteins can be achieved in reverse phase arrays using recombinant proteins derived from transient transfectants in a non-purified form. We also used forward phase arrays for competitive analysis of antibodies and mapping of their epitopes. This approach is valuable for rapidly screening antibody specificities and assessing protein orientation needed for optimal presentation of immunogenic determinants.
We also showed that binding of CD200 ligand to its cell surface receptor can be achieved by increasing the avidity of the reaction via coupling of the biotinylated recombinant CD200 protein to fluorescently labelled avidin coated beads. The fluorescent beads offer an efficient technology for the analysis of low affinity interactions typical of those observed for leukocyte membrane proteins and many other cellular proteins.
Monoclonal antibodies (mAb) DX147 (rat IgG1), and DX136 (rat IgG2a) to human CD200R were generously given by DNAX Research Institute (Palo Alto, CA). The mAb OX108 (mouse IgG1) to human CD200R  and OX68 (mouse IgG1) to rat CD4 domains 3 and 4 (rCD4d3+4) have been described previously .
The soluble biotinylated forms of human, mouse and rat CD200R were produced as described [22, 31, 36]. Briefly, the entire extracellular region of human, mouse or rat CD200R was amplified by PCR and cloned in the pEF-BOS-CD4d3+4bio-XB vector . These constructs were then subcloned into the expression vector pEE14, and stably secreting CHO.K1 cell lines were established . Human, mouse or rat CD200R-CD4d3+4 proteins were purified from the tissue culture medium by immunoaffinity chromatography with OX68 mAb-Sepharose 4B that recognizes the CD4 protein tag . Prior to use, the purified CD200R proteins were fractionated by gel filtration on Superdex S-200 (Pharmacia, Uppsala, Sweden) to exclude protein aggregates. The soluble, biotinylated form of human CD200 was produced in an identical fashion, by subcloning the amplified extracellular region of human CD200  using Xba I/Sal I digestion to the pEF-BOS-CD4d3+4bio-XB vector . This construct was used to transfect HEK293T cells using the calcium phosphate method. The protein expressed was enzymatically biotinylated and used to generate multivalent binding reagents by coupling to avidin-coated fluorescein isothiocyanate (FITC)-loaded beads (Spherotech Inc., Libertyville, IL) as described previously .
Mutants of human CD200R (hCD200R) were prepared as described . Briefly, the mutations were introduced by site directed mutagenesis using PCR and two mutagenic oligonucleotides into a construct comprising the extracellular domains of human CD200R together with domains 3 and 4 from rat CD4 (CD4d3+4) as an antigenic tag. The mutants were transiently expressed in HEK 293T cells using X-VIVO 10 media (BioWhittaker, Nottingham), concentrated about 10 fold and levels of expressed protein quantified by ELISA. This media contains 1 mg ml-1 BSA so after concentration the final protein concentration is around 10 mg/ml.
Purified antibodies were dialysed against PBS prior to labelling with Alexa Fluor 555 or Alexa Fluor 647 fluorescent amine-reactive dyes using the Molecular Probes Monoclonal Antibody Labelling Kits (Cat. No. A-20186 and A-20187) and according to the manufacturer's instructions (Molecular Probes, Invitrogen Ltd.). Labelling reactions were carried out using 100 μg of IgG and yielded labelled proteins ranging in concentration from 1 to 4 × 10-6 M. The degree of conjugation was estimated at 2–4 moles of dye per mole protein. Labelled antibodies were stable for up to 2 months at 4°C.
Preparation of microarrays
Protein solutions to be arrayed were prepared in 96 well plates and 12 μl aliquots were transferred to single wells of Genetix 7020, 384-well plates (Genetix Ltd, New Milton, UK). Concentrations tested ranged from 0–80 μg/ml and all dilutions were performed in Protein Array Spotting Solution (Genetix) with the addition of 0.5 mg/ml BSA and 0.02% NaN3. A QArrayMini microarray printer (Genetix) was used to apply the protein solutions onto epoxy-coated microscope slides using 300 μm solid tipped tungsten microarraying pins (Genetix). Preliminary experiments established the printing conditions with fluorescently labeled OX68 mAb. Of several types of slides tested, the epoxy-coated ones were the best in terms of spot morphology, cost and reproducibility and were used in all subsequent experiments.
Most array designs were performed using 8 pins to obtain spots with a 440 μm diameter and centre-to-centre spot spacing of 700 μm in both directions. Source plates were kept at 8°C, and a 65% average internal humidity was maintained. After printing, the slides were left in the arraying chamber for 30–60 minutes under the same conditions. The slides were then washed using the Protein Array Processing Kit (Genetix Ltd; stored at 4°C and the solutions supplemented with 0.02 % NaN3) by inversion for 1 min in Clean Up Buffer (Genetix) to remove unbound proteins and incubation for 30 min in Blocking Buffer. Slides were washed 3 times in PBS, once in H2O to remove excess salt and dried using an air brush, and stored at 4°C, with desiccant in a sealed slide box. Preliminary experiments were done by forward phase arrays to establish optimal conditions. OX68 mAb (100 μg ml-1 to 20 μg ml-1) was immobilized, incubated with rat CD4 at 5 μg ml-1 0.1 mg ml-1 BSA, washed and incubated with labeled W3/25 mAb (5 μg/ml) and linearity of detection demonstrated. Specificity was also shown by the fact that OX68 was not reactive with CD4 immobilised on OX68 and vice versa.
Labelling of microarrays
Slides were placed in hybridization chambers (Corning Incorporated, UK) and the humidification wells filled. LifterSlips, (Erie Scientific, Portsmouth, USA) were placed gently over the marked boundaries of the arrays and the binding reagent (25–70 μl) was introduced with a micropipette. In experiments where CD200R-CD4 hybrid proteins (including the mutant studies) were arrayed, Alexa-555 anti-CD200R antibodies (mAb DX147, DX136 or OX108; 5–10 μg ml-1) were added to measure the amount of specific antibody bound and Alexa-647-CD4 mAb (OX68 5–10 μg ml-1) was included to assess the amount of hybrid protein present. In experiments where capture antibodies were arrayed (Fig. 2), a 2 h incubation with purified protein, such as CD200R-CD4d3+4 or CD4d3+4 at 20 μg ml-1, was performed prior to incubation with the detection antibodies. In the experiments detecting CD200 (ligand) binding to immobilized CD200R (Fig. 5), arrays were incubated with biotinylated hCD200-CD4d3+4 streptavidin-FITC beads. Incubations with detection reagents were carried out for 16 h at 4°C unless otherwise stated. The slides were immersed upside down in PBS/0.05% Tween-20, washed thrice with copious amounts of PBS/Tween-20, alternating shaking up and down under liquid in the Copeland jar and gentle rocking for 5–10 min each, followed by PBS (twice for 5 min) and a final H2O rinse. All washes were at room temperature and repeated following each incubation. After drying the slides with an air brush, the arrays were scanned using a GenePix4000B microarray scanner (Axon Laboratories, Palo Alto, CA) scanner using 532 nm and 635 nm lasers using the GenePix Pro 5.0 (Axon Laboratories) software. The PMT values were 720 and 1000 (532 nm and 635 nm respectively) for Figure 1, 780 and 950 for Figure 2, 900 and 1000 for Figure 3 and 850 for Figure 5 (532 nm only).
All samples were tested in quadruplicate and all experiments repeated several times. The amount of antibody or ligand bound to the arrayed proteins and the amount of protein present in each spot were determined by comparing the fluorescence intensities read at 532 and 635 nm. Extraction of spot intensity data was performed using GenePix Pro 5.0 (Axon Laboratories) and ScanArray Express (Perkin Elmer). The background, calculated as the median of pixel intensities from the local area around each spot, was subtracted from the mean pixel intensity within each spot. To graphically represent the data, the values of the background-subtracted signal intensities were plotted against the known concentration of the protein spotted in the array. Sensitivity of detection for each spot was defined as a signal to noise ratio (S/N) of two-fold above background. S/N was calculated as: S/N = (background-subtracted median signal intensity) / (standard deviation of background signal intensity).
In the case of the mutant hCD200R proteins generated from culture supernatants of transient transfections, where protein concentration is unknown, the background-subtracted values for both 532 and 635 nm-signal intensities were corrected for internal protein signal by subtracting the corresponding value of a "mock transfectant" spot. The corrected values for the red channel (representing the amount of protein assessed from the CD4 content) were normalised to 100% with respect to the wild-type hCD200R transient transfection sample. All hCD200R mutants with red channel values below 50% were assumed to contain insufficient amount of protein and were excluded from the analysis. The green channel background-subtracted, "mock"- transfectant corrected values (G) were divided by the corresponding red channel ones (R) to correct for variations in the amount of expressed protein contained in each individual spot (G/R ratio). Finally, the G/R ratio was normalized to 100% with respect to the hCD200R-CD4d3+4 wild-type protein, before graphical representation.
List of abbreviations
chimaeric recombinant CD200 protein with rat CD4 domains 3+4
human CD200 receptor
mouse CD200 receptor
rat CD200 receptor
surface plasmon resonance
This work was supported by the Medical Research Council (UK) and the Arthritis Research Campaign (UK). The microarray infrastructure established with support from the EPA Cephalosporin Trust. We are grateful to Holly Cherwinski and Joe Phillips (DNAX, Palo Alto) for the gift of purified DX147 and DX136 mAb and advice.
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